Determination of amylase

ABSTRACT

A method for the determination of amylase activity and a useful highly stable color reagent aqueous solution therefore are disclosed. The solution comprises, as a color reagent, an aromatic nitro containing compound such as 3,5-dinitrosalicyic acid, a color stabilizing chelating compound such as ethylenediaminetetraacetic acid, and a hydroxide base. Eliminating the necessity for centrifuging prior to the determination of amylase activity can be achieved by including potassium hydroxide for at least a part of the base in the solution.

llnite 1 Mar. 4, 1975 DETERMINATION OF AMYLASE [75] Inventor: E. Melvin Gindler; Rockford, Ill.

[73] Assignee: Pierce Chemical Company,

Rockford, Ill.

22 Filed: Feb. 26, 1973 [21] Appl. No.: 335,736

[52] US. Cl. 195/99, 195/1035 R [51] Int. Cl. G0ln 31/14, GOln 33/16 [58] Field of Search 195/1035 Searcy et aL, The America] Journal of Clinical Pathology Vol. 46 No. 5 pages 582-586 (1966).

Primary ExaminerAlvin E. Tanenholtz Attorney, Agent, or Firm-W0lfe, Hubbard, Leydig, Voit & Osann,

[57] ABSTRACT A method for the determination of amylase activity and a useful highly stable color reagent aqueous solution therefore are disclosed. The solution comprises, as a color reagent, an aromatic nitro containing compound such as 3,5-dinitrosalicyic acid, a color stabilizing chelating compound such as ethylenediaminetetraacetic acid, and a hydroxide base. Eliminating the necessity for centrifuging prior to the determination of amylase activity can be achieved by including potassium hydroxide for at least a part of the base in the solution.

10 Claims, N0 Drawings DETERMINATION OF AMYLASE The present invention relates to the determination of amylase activity. More particularly, it relates to an improved saccha'rogenic procedure for the determination of amylase.

Amylase is known to catalyze the hydrolysis to starch resulting in the formation of lower glucose polymers such as maltose, trioses, etc., hereafter referred to as lower sugars. It is known that the lower sugars can, under appropriate conditions, reduce certain organic compounds. Thus, by using an organic compound which exhibits a color change on reduction by products resulting from the hydrolysis of starch, customary colorimetric techniques can be used to indicate the degree to which starch hydrolysis has occurred and, in turn,

the quantity of amylase originally present. The determination of amylase is medically important since the presence of high levels of amylase in blood serum or other biologic fluids may indicate pancreatitis.

A saccharogenic procedure which follows the rate of lower sugar production after the addition of a serum sample to a starch substrate has been frequently employed in determining amylase activity. In this procedure, the color reagent (reducible by the lower sugars) is added to the starch-serum mixture after a predetermined amount of time. Addition of the color reagent terminates the enzyme catalyzed hydrolysis of starch and, thereafter, the degree of hydrolysis which has been achieved is indicated by the extentto which the added quantity of color reagent is reduced by the lower sugars produced. Amyloclastic methods, which follow the rate at which starch or turbidity of a starch-serum mixture disappears, are also used for indicating amylase activity. However, it is believed that the present invention is principally applicable to saccharogenic procedures.

The use of dinitrosalicylic acid (DNSA) for the determination of reducing sugar has been known for many years (e.g., Sumner and Graham, J. Biol. Chem. 47, (1921 and Miller, Analytical Chemistry, Vol.31, 426-428 (1959)). The use of DNSA to saccharogenically determine amylase activity is also known (e.g., Searcy et al., The American Journal ofClinical Pathology, 46, 582-586 (1966)). Generally, amylase activity is reported in Somogyi units. By definition, a Somogyi unit of amylase activity equals 0.01 mg of reducing substance, expressed as glucose, generated by incubating 1.0 ml of serum according to the Somogyi procedure which is described in J. Biol. Chem. 125, 399 (1938).

Concerning the use of DNSA, Miller, supra, indicates that for the determination of reducing sugar, ingredients other than DNSA are necessary-These include Rochelle salt (potassium sodium tartrate) to prevent dissolution ofoxygen, phenol to increase the amount of color produced and to balance the effect of phenol encountered in urine, sodium bisuliite to stablize the color obtained in the presence of phenol, and sodium hydroxide to permit the reducing action of glucose on DNSA. The article indicates that the use of the R0- chelle salt, while being essential to color stability, inter- Searcy et al., supra, appears to describe a procedure using DNSA color reagent, sodium hydroxide and Rochelle salt without the presence of phenol and sulfite and states that the reagent is stable for at least six months when stored in a tightly sealed amber bottle to prevent undue exposure to light and air. The absence of the bisulfite is believed to result in an inability to indicate small levels of amylase particularly with starch substrates which have been pretreated to remove reducing groups such as aldehydes.

A procedure using an essentially non-reducing starch substrate has the advantage that small errors in substrate volume produce virtually insignificant error in the final result. Also, many colorimeters are not accurate nor can be set at absorbance 0 with reagent blanks of high absorbance and reducing starches tend to give such high absorbance. However, an initial disadvantage of using non-reducing starches is that the straight graph lines (glucose v. absorbance) do not go through the origin; a very common problem with 3,5- dinitrosalicylic acid. The addition of phenol and sulfite to the system causes the straight graph lines to approach more closely the origin, thereby enhancing the ability to detect low levels of reducing sugar and, in turn, low concentrations of amylase.

In its broadest aspects, it is an object of the present invention to provide an improved procedure for determining amylase activity which realizes the benefits of using reducibile color reagents such as DNSA but in which the presence of Rochelle salt with its attendant problems is not necessary in order to achieve color stability. More particularly, an object of the invention resides in providing a highly stable color reagent solution which is effective in indicating small levels of amylase concentration in biologic fluids. A more specific object resides in providing a lower sugar reducible reagent solution including sulfite ions to permit effective measurements of small concentrations of amylase and wherein the reagent solution is itself stable for extended periods of time. Yet a further object is to provide a reagent solution wherein the subsequently formed reduced solution has a color which is stable for many hours.

As indicated above, the presence of phenol is also desirable in procedures using reducible color reagents. However, phenol is unstable in the presence of air, poisonous, and exhibits an unpleasant odor. Accordingly, a further object of the present invention is to provide a color reagent solution which is sensitive in detecting amylase but wherein phenol is not present.

Referring again to procedures using DNSA, it is generally necessary to heat the solution formed after the addition of DNSA to the hydrolyzed starch substrate in order to effect reduction. It has been noted that upon subsequent cooling when a biologic fluid such as blood serum is present, particles sometimes appear creating turbidity and necessitating centrifuging before spectrophotometric analysis. Thus, a further object ofthe present invention is to provide a procedure and, in turn, an improved color reagent solution, wherein particles are not formed in significant quantity and the necessity for centrifuging is obviated.

Briefly stated, all ofthe objects and aims of the present invention can be realized by using a reducible color reagent solution which contains, in addition to the color reagent itself, ethylenediaminetetraacetic acid (EDTA), sodium salicylate, sodium sulfite, sodium hytlroxide, and potassium hydroxide. Surprisingly, the use of EDTA, instead of the Rochelle salt, has been found effective in providing a highly color stable solution both as prepared and after additionto the hydrolyzed starch substrate. There is no apparent interference with the'protective action of the sulfite. In addition, EDTA is believed to effectively chelate multivaliant metal ions which may be present in the serum and thus prevent them from precipitating or otherwise interferring. The sodium salicylate appears to enhance the sensitivity of the measurement, but its use is not accompanied by those disadvantages identified above associated with the presence of phenol. Lastly, the presence of potassium hydroxide eliminates the problem of turbidity after heating and thus centrifuging is not necessary.

'The following example illustrates the present inventton.

PREPARATION OF STARCH SUBSTRATE Twenty grams of soluble potato starch is dissolved in 400 ml deionized water with heat and stirring. The solution is cooled to room temperature and 0.60 grams of KBH is added and the solution stirred for 2 hours at room temperature. Then 1.6 ml of acetone are added and the solution stirred for an additional 20 minutes. As described by Strumeyer,-Analytical Biochemistry 19, 6l-7l (1967), treatment with borohydride removes aldehyde groups their precursors by converting the terminal glucosyl groups in the starch to noninterferring glucitol residues.

The starch solution so prepared is then diluted with water to about 1.9 liters and the following ingredients which are a combination of preservatives, accelerators and buffers are added while the solution is stirred.

5.0 ml. 37% HCHO (Reagent) 4.0 grams Potassium Sorbate 7.50 grams KH PO 8.00 gm. Na HPO 8.0 grams NaCl 4.0 grams NaF The total volume of the resulting solution is then atljusted to 2 liters by addition of deionized water and when stored at room temperature is stable for at least about 6 months.

PREPARATION OF DNSA COLOR REAGENT SOLUTION The following reagents are added to water to give 1 liter of solution with the DNSA being added first and L85 gm. 3.5-Dinitrosalicylic Acid H 1.5 gm. Na H EDTA '2H O (Dihydrate of dlsodium salt of ethylenediaminetetraacetic acid) 20 ml. 19 M-NaOH 40 ml. 45% potassium hydroxide Add to increase ionic strength to a high level and thus prevent significant ionic strength variations.

AMYLASE DETERMINATION Two 13 X 100 mm test tubes, one marked T (test) and the other 8" (blank) are employed using the following procedures. Control serum of high amylase activity (such as Warner-Chilcott Versatol E" or Dade Moni-trol ll or Enza-trol") is used as the standard.

Normal (isotonic) saline (e.g., the sodium chloride solution intended for injection) solution can be used to prepare dilutions of the serum, for the preparation of a colorimetric calibration graph (absorbance versus amylase activity). Saccharogenic procedure values given with the control serum are used. An alternative standard is serum previously analyzed by means of a reference procedure. A further alternative standard is a solution of glucose which has been compared with serum of known amylase activity. lt is to be understood that glucose solution essentially evaluates only the color reagent.

T-Tube (Test) B-Tubc (Blank) l. Add 0.050 ml. microliters) l.

serum.

2. Add 0.50 ml. Substrate,

Add 0.050 ml. (50 microliters) serum.

Add 0.50 ml DNSA reagent. Mix well.

pre-warmed at 37C. mix well and place in water bath at 37C. 3. Add 0.50 ml. substrate. 3. Exactly 15 minutes after adding Mix well.

substrate, add 0.50 ml. DNSA 4. Heat I0 minutes at C. reagent. Mix well. 5. Cool in cold water bath for 4. Heat 10 minutes at l00C. 3 minutes. 5. Cool in cold water bath for 3 6. Add 4.00 ml. distilled minutes. water. Mix well. 6. Add 4.00 ml. distilled water. 7. Set absorbance at 0 in a Mix well.

colorimeter or spectrophotometer at 500 nm.

values.

Amylase Activity of Sample, in Somogyi Units /1 00 m1.

(Absorbance of Sample versus its Blank) SX Absorbance of Standard versus its Blank) S given amylase activity of standard in Somogyi Units/100 m1.

1,2-Dinitrobenzene;

If a graph is not linear then the amylase activity is read from the graph. Serum amylase activity in excess of 120 Somogyi Units/100 ml. may indicate pancreatitis.

While there has above been described in detail a pre ferred embodiment of the present invention, it should be understood that the present invention is susceptible of various modifications. It is not intended to limit the invention to the specific embodiments theretofore disclosed. On the contrary, the inventionis to cover all modifications and alternatives falling within the spirit and scope of the invention as expressed in the appended claims.

For example, while the invention has been illustrated with respect to DNSA color reagent, the use of which is desirable due to the virtual absence of protein interference, the advantages achieved thereby are believed to be equally applicable to the other compounds reducible by the lower sugars which, in their reduced condition, exhibit a different color or intensity than in their unreduced form. The compounds, of course, should not be so easily reducible that they are reduced by EDTA or other ingredients present. Aromatic compounds containing a nitro group reducible to an amine or other colored substance are believed to be useful. Examples of such compounds include the following:

3,4-Dinitrobenzoic Acid; 3,6- Dinitrophthalic Acid; 3,5-Dinitrosalicylic Acid (DNSA); 2,4,6-Trinitro-phenol (Picric Acid); 2,4- Dinitro-phenol; and 5,7-Dinitro-8- hydroxynaphthalene-Z-sulfonate. Where protein exhibits a significant reducing effect on the compound, deproteinization should be accomplished prior to analy- SIS.

It will also be appreciated that various other chelating compounds such as other derivatives of iminodiacetic acid can be substituted for all or part of the specially illustrated EDTA. EDTA has the following structure CH2 N tca coorn (II-12* N (CH2COOH)2 and examples of other useful compounds include diethylene triamine pentacetic acid and ethylenebis (oxyethylene nitrilo) tetraacetic acid. EDTA is generally available as any ofseveral salts or the free acid as well as hydrated and it can be used in any of these forms. Of

course, the compounds selected should not contain groups having reducing properties such as aldehyde groups.

The amounts of color reagent andchelating compound employed in accomplishing the present invention are not particularly critical. For example, DNSA can be employed in about 0.1 0.3 grams/100 ml. of reagent solution, and, preferably, 0.15 0.20 gram. Similarly, on the same basis, EDTA is useful in an amount of about 0.1 0.4 grams and, preferably, 0.13 0.17 grams. It should be noted that these amounts of EDTA are considerably less than the quantity of R0- chelle salt needed. The sulfite and salicylate are ordinarily used in about equal amounts of about l-4 grams/100 ml., respectively. Other sulfite and salicylate salts, e.g., potassium, can be used in molarities substantially equivalent to the illustrated sodium salts. Regarding the quantity ofsodium hydroxide and potassium hydroxide employed, it is necessary that a sufficient quantity ofthese bases be employed to elevate the pH of the solution to a value where the reducing effect of the lower sugars on the color reagent is enhanced. Typically, the pH should be above about 10 and preferably above 12. Accordingly, the combined amounts of sodium and potassium hydroxide should be greater than about 0.001 mole/ ml.

However, while the total amount of hydroxide present is not particularly critical, it is an important aspect of the present invention that the molar ratio of the potassium hydroxide to sodium hydroxide be at least about 1 and preferably at least about 1.2 in order to avoid the appearance of particles as discussed above with substantially all types of human serum. While there is no upper limit on the ratio of potassium hydroxide to sodium hydroxide and there is no necessity for sodium hydroxide being present at all, from a cost point of view it is generally-desirable to use the minimum amount of potassium hydroxide necessary to prevent particle formation and provide a color stable solution.

While the invention has been illustrated with the direct use of potassium hydroxide, :it is believed that it is the presence of the potassium ion which prevents particle formation. Consequently, if potassium salts such as of the carbonate, sulfite. salicylate, etc. are used, the addition of potassium hydroxide can possibly be reduced or eliminated. In any event, it is believed that potassium ion concentration in the solution must be at least about 0.03 mole/100 ml. of solution and preferably at least about 0.04 mole. In addition to potassium other higher alkali metal cations {e.g., Rb, Cs, Fr) are thought to be useful but are economically unattractive.

I claim as my invention:

1. An aqueous solution useful for the detection of amylase activity comprising an aromatic nitro containing compound reducible by lower sugars to give a re duced compound having a different color or color intensity, a color stabilizer, sufficient base to raise the pH above about 10, and at least about 0.03 mole/100 ml of a higher alkali metal cation, said color stabilizer being an iminodiacetic acid containing chelating compound having substantially no reducing properties.

2. The aqueous solution of claim 1 wherein the aromatic nitro containing compound is 1.2- dinitrobenzene; 3,4-dinitrobenzoic acid; 3.6- dinitrophthalic acid; 3.5-dinitrosalicylic acid: 2.4.61- trinitro-phenol; 2,4-dinitrophenol; or 5.7-dinitro-8- hydroxynaphthaleneQ-sulfonate.

3. The aqueous solution of claim 2 wherein the aromatic nitro containing compound is 3,5-dinitrosalicylic acid.

4. The aqueous solution of claim 1 wherein the color stabilizer is ethylene diaminetetraacetic acid, diethylene triamine pentacetic acid or ethylenebis oxyethylene nitrilo tetraacetic acid.

5. The aqueous solution of claim 4 wherein the color stabilizer is ethylene diaminetetraacetic acid.

6. The aqueous solution of claim 1 containing at least about 0.04 mol./10O ml. of potassium ion.

7. The aqueous solution of claim 3 wherein the color stabilizer is ethylene diaminetetraacetic acid.

8. The aqueous solution of claim 7 containing potassium ions in a concentration of at least 0.04 mol./100 ml. of solution and wherein the molar ratio of potassium ion to any sodium ion present is at least about 1. 10. The composition of claim 9 containing, in addi- 9. The aqueous solution of claim 8 wherein the molar tion to the ingredients included therein, salicylate ions,

ratio of potassium ion to sodium ion, if present, is at sulfite ions, carbonate ions and hydroxyl ions.

least about 1.2. 

1. AN AQUEOUS SOLUTION USEFUL FOR THE DETECTION OF AMYLASE ACTIVITY COMPRISING AN AROMATIC NITRO CONTAINING COMPOUND REDUCIBLE BY LOWER SUGARS TO GIVE A REDUCED COMPOUND HAVING A DIFFERENT COLOR OR COLOR INTENSITY, A COLOR STABILIZER, SUFFICIENT BASE TO RAISE THE PH ABOVE ABOUT 10, AND AT LEAST ABOUT 0.03 MOLE/100 ML OF A HIGHER ALKALI METAL CATION, SAID COLOR STABILIZER BEING AN IMINODIACETIC ACID CONTAINING CHELATING COMPOUND HAVING SUBSTANTIALLY NO REDUCING PROPERTIES.
 2. The aqueous solution of claim 1 wherein the aromatic nitro containing compound is 1,2-dinitrobenzene; 3,4-dinitrobenzoic acid; 3,6-dinitrophthalic acid; 3,5-dinitrosalicylic acid; 2,4,6-trinitro-phenol; 2,4-dinitrophenol; or 5,7-dinitro-8-hydroxynaphthalene-2-sulfonate.
 3. The aqueous solution of claim 2 wherein the aromatic nitro containing compound is 3,5-dinitrosalicylic acid.
 4. The aqueous solution of claim 1 wherein the color stabilizer is ethylene diaminetetraacetic acid, diethylene triamine pentacetic acid or ethylenebis oxyethylene nitrilo tetraacetic acid.
 5. The aqueous solution of claim 4 wherein the color stabilizer is ethylene diaminetetraacetic acid.
 6. The aqueous solution of claim 1 containing at least about 0.04 mol./100 ml. of potassium ion.
 7. The aqueous solution of claim 3 wherein the color stabilizer is ethylene diaminetetraacetic acid.
 8. The aqueous solution of claim 7 containing potassium ions in a coNcentration of at least 0.04 mol./100 ml. of solution and wherein the molar ratio of potassium ion to any sodium ion present is at least about
 1. 9. The aqueous solution of claim 8 wherein the molar ratio of potassium ion to sodium ion, if present, is at least about 1.2.
 10. The composition of claim 9 containing, in addition to the ingredients included therein, salicylate ions, sulfite ions, carbonate ions and hydroxyl ions. 